Superior RNA for Demanding Applications

Obtaining high quality RNA, especially human RNA, can be challenging because of factors that are often difficult to control. Tissue procurement is probably the biggest hurdle for many sample types. First a reputable and ethical tissue source must be identified. Typically you then wait for the desired tissue type and sample size to become available, and finally you hope that the tissue was harvested quickly and handled properly to avoid RNA degradation. Once you have obtained the desired tissue sample, it must be disrupted and homogenized using the technique and reagents best suited to the tissue source. After completing the RNA isolation procedure, it is also crucial for demanding applications to assess the quantity, quality, and purity of the RNA. For successful gene expression analysis and cloning experiments, accurately quantitated, full length, pure RNA is required. Many applications also demand that the RNA be purified of contaminants such as genomic DNA, ribosomal RNA (in mRNA preps), protein, ethanol, organic solvents, and salt.

Ambion's prepared RNAs are available for researchers who choose to avoid these challenges. Our FirstChoice® RNAs are available from a variety of human, mouse, and rat tissues, and from cell lines. The FirstChoice RNAs, which are stored in THE RNA Storage Solution to promote long term stability, pass the industry's most stringent quality control tests. The chart in Figure 1 describes the different RNA products available and the applications for which they are recommended.

Product	Description	Size	Quality Standards	Recommended Applications
FirstChoice® Total RNA	Total RNA from specific human, mouse and rat tissues, human brain regions, human and Drosophila cell lines and E.coli*	Human Tissues & Cell Line- 100 µg Brain Region- 50 µg Mouse & Rat- 200 µg E.coli- 200 µg	Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR	RT-PCR, Array Analysis, Northern Analysis, RPA, cDNA Library Construction
FirstChoice® Poly(A) RNA	Poly(A) RNA from specific human, mouse and rat tissues and human and Drosophila cell lines*	5 µg	Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR, mRNA enrichment determined by Northern analysis	RT-PCR, Array Analysis, Northern Analysis, RPA, cDNA Library Construction
FirstChoice® Tumor/Normal Adjacent Tissue RNA	Total RNA from pairs of individual human tumor and normal adjacent tissues*	5 µg of each	Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR	RT-PCR, Array Analysis, RPA cDNA Library Construction
FirstChoice® Tumor RNA	Total RNA from individual human tumors*	5 µg	Quality and stability checked using the Agilent 2100 bioanalyzer, tested for removal of DNA by real-time RT-PCR	RT-PCR, Array Analysis, RPA cDNA Library Construction
*All human RNAs include donor demographic information and, when applicable, pathology/histology information.

Figure 1. Description of Ambion's High Quality RNAs.

Ambion now offers high quality FirstChoice Total RNA from individual regions of the human brain. Traditionally, it has been difficult for researchers to obtain high quality RNA from human brain tissue. Ambion has established relationships within the research and medical community to ethically obtain tissues in a manner that preserves the integrity of the RNA. We have also optimized RNA isolation procedures specifically for brain and neural tissue. These efforts, in conjunction with stringent quality control standards, ensure that FirstChoice Brain Total RNA is intact, stable, and free of genomic DNA (<0.05%). Results from one of our Quality Control assays are shown in Figure 2. 



Replicate samples of total brain RNA were stored overnight at either 20ºC or at 37ºC and then analyzed by capillary electrophoresis using the Agilent 2100 bioanalyzer. Even after overnight incubation at 37ºC, the ratio of the areas of the peaks representing 28S and 18S rRNA are approximately 2:1; this illustrates the high quality and stability of FirstChoice RNA. Total RNA is currently available from the following regions of the brain: frontal cortex, occipital cortex, pariental cortex, temporal cortex, basal ganglia, cerebellum, medulla, and thalamus.

FirstChoice Total RNA is available from a wide variety of human, mouse, and rat tissues and cell lines. The samples are processed using Ambion's RNA isolation reagents to recover highly pure, intact RNA. A stringent DNase treatment is included to destroy any contaminating genomic DNA, and to ensure that the RNA is ready for use in downstream applications such as RT-PCR. Although it is impossible to remove every trace of DNA contamination from an RNA sample, FirstChoice RNAs are tested by real-time PCR to ensure that residual DNA contamination is insignificant (<0.05%). Additionally, the RNAs undergo rigorous testing for nucleases. The integrity and stability of each RNA is verified by capillary electrophoresis using the Agilent 2100 bioanalyzer as shown in Figure 2.

FirstChoice Poly(A) RNA is isolated from the FirstChoice Total RNA described above. The RNA undergoes poly(A) selection using Ambion's Poly(A)Purist™ technology; a method that outperforms other purification methods and yields RNA that is more highly enriched for poly(A) RNA and is nearly devoid of rRNA. Because of the high quality of starting material (FirstChoice Total RNA), and the unsurpassed level of mRNA enrichment, 5 µg of Ambion's FirstChoice Poly(A) RNA contains more mRNA than other commercially available poly(A) RNA preparations which are often highly contaminated with rRNA, DNA or both. An electropherogram of FirstChoice Poly(A) RNA is shown in Figure 4; it illustrates the purity of the preparation.

Also part of the FirstChoice line of products, the FirstChoice Tumor and FirstChoice Tumor/Normal Adjacent Tissue total RNAs are prepared using the same methods and quality standards as the FirstChoice Total RNAs, but are derived from human tumor samples. The FirstChoice Tumor RNAs include 5 µg of total RNA from an individual human tumor. The FirstChoice Tumor/Normal Adjacent Tissue RNA pairs include 5 µg each of total RNA isolated from an individual tumor and the normal adjacent tissue (NAT).

All human tissues used for RNA isolation at Ambion are obtained through accredited organ/tissue procurement organizations that operate with Institutional Review Board approval, and in accordance with regulations set forth by the United States Department of Health and Human Services. These regulations include donor consent and protection of privacy regulations.